Kinetoplast DNA is the mitochondrial DNA of trypanosomes and related parasitic protozoa. It is an unusual DNA, consisting of DNA circles that are catenated into a giant network. The network contains several thousand minicircles and a few dozen maxicircles. The subject of this proposal concerns the structural organization and the replication of kDNA. Major steps in kDNA synthesis include the sequential release of minicircles from the network by a topoisomerase to form free minicircles, replication of the free minicircles via theta structures, and reattachment of the progeny minicircles to the network. An interesting feature of the kDNA replication system is that replication enzymes are localized in specific sites around the kDNA in vivo. Since there is no structure resembling kDNA in humans, the host for these parasites, an understanding of kDNA replication might suggest some novel approaches to selective anti-parasite chemotherapy. One major aim of this proposal is to purify and study enzymes involved in kDNA replication in Crithidia fasciculata, with a major focus on the replicative DNA polymerase and the DNA primase. The primase is of special interest because it appears to contain an RNA component that may be essential for enzymatic activity. A second aim is to study the mechanism of minicircle replication, and in these experiments there will be a major emphasis on the intramitochondrial localization of minicircle replication intermediates. These experiments will depend heavily upon fluorescence in situ hybridization (FISH). The third aim will be to study the mode of reattachment of newly synthesized minicircles to the Trypanosoma brucei network, using both fluorescence microscopy and autoradiography at the electron microscope level. The final aim will be to study DNA repair in the mitochondrion of Crithidia fasciculata.